Toxicon 108 (2015) 141e146 Contents lists available at ScienceDirect Toxicon journal homepage: www.elsevier.com/locate/toxicon Toxic Takifugu pardalis eggs found in Takifugu niphobles gut: Implications for TTX accumulation in the pufferfish * Shiro Itoi a, , Ao Kozaki a, Keitaro Komori a, Tadasuke Tsunashima a, Shunsuke Noguchi a, 1, Mitsuo Kawane b, Haruo Sugita a a Department of Marine Science and Resources, Nihon University, Fujisawa, Kanagawa 252-0880, Japan b Department of Sea-Farming, Aichi Fish Farming Institute, Tahara, Aichi 441-3618, Japan article info abstract Article history: Pufferfish (Takifugu spp.) possess a potent neurotoxin, tetrodotoxin (TTX). TTX has been detected in Received 9 July 2015 various organisms including food animals of pufferfish, and TTX-producing bacteria have been isolated Received in revised form from these animals. TTX in marine pufferfish accumulates in the pufferfish via the food web starting with 30 September 2015 marine bacteria. However, such accumulation is unlikely to account for the amount of TTX in the puf- Accepted 14 October 2015 ferfish body because of the minute amounts of TTX produced by marine bacteria. Therefore, the tox- Available online 19 October 2015 ification process in pufferfish still remains unclear. In this article we report the presence of numerous Takifugu pardalis eggs in the intestinal contents of another pufferfish, Takifugu niphobles. The identity of Keywords: Congeneric eggs T. pardalis being determined by direct sequencing for mitochondrial DNA. LC-MS/MS analysis revealed fi Food chain that the peak detected in the egg samples corresponded to TTX. Toxi cation experiments in recirculating Pufferfish aquaria demonstrated that cultured Takifugu rubripes quickly became toxic upon being fed toxic (TTX- Takifugu niphobles containing) T. rubripes eggs. These results suggest that T. niphobles ingested the toxic eggs of another Takifugu pardalis pufferfish T. pardalis to toxify themselves more efficiently via a TTX loop consisting of TTX-bearing or- Tetrodotoxin ganisms at a higher trophic level in the food web. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction pufferfish and the potential food organisms (Noguchi et al., 1987; Wu et al., 2005; Noguchi and Arakawa, 2008). In addition, TTX Tetrodotoxin (TTX) is known to be the substance of pufferfish has been detected in some free-living bacteria, including those in toxin and one type of potent neurotoxin specific to voltage-gated deep sea sediments (Simidu et al., 1987; Do et al., 1990), although it sodium channels of excitable membranes of muscle and nerve is not clear if these bacteria form part of the food chain leading to tissues (Colquhon et al., 1972; Narahashi, 2001; Noguchi et al., pufferfish. In any case, it appears plausible that the TTX in pufferfish 2006a). TTX was believed to occur only in pufferfish (Tetraodonti- is a result of accumulation through the food chain, which consists of dae) until 1960's, when it was detected in Californian newt Taricha several steps, starting with bacteria, as suggested by several reports torosa (Mosher et al., 1964). Subsequently, TTX (along with some (Noguchi et al., 2006a; Noguchi and Arakawa, 2008). These spec- analogs) was also detected from potential pufferfish food organ- ulations have actually been supported by several studies: non-toxic isms belonging to various disparate groups, including starfish (e.g., pufferfish have been produced when grown from hatching with a Astropecten spp.; Maruyama et al., 1984, 1985), gastropods (e.g., non-toxic diet, and furthermore, these cultured non-toxic puffer- Babylonia japonica; Noguchi et al., 1981), crustaceans (e.g., the fish have become toxic when administered orally with TTX (Matsui xanthid crab, Atergatis floridus; Noguchi et al., 1983), flatworms and et al., 1981, 1982; Noguchi et al., 2006b; Saito et al., 1984; Yamamori ribbonworms (e.g., Cephalothrix simula; Asakawa et al., 2013), apart et al., 2004; Honda et al., 2005). from several species of bacteria that are symbiotic with the TTX has been detected not only in pufferfish and their prey, but also in organisms ecologically unrelated to pufferfish, such as the Costa Rican frogs of the genus Atelopus (Kim et al., 1975) and some * Corresponding author. land planarians (Stokes et al., 2014), besides Californian newt E-mail address: [email protected] (S. Itoi). T. torosa (Mosher et al., 1964). It has also been suggested that TTX in 1 Present address: Kyoto Institute of Oceanic and Fishery Science, Miyazu, Kyoto the rough-skin newt, Taricha granulosa, is obtained endogenously 626-0052, Japan. http://dx.doi.org/10.1016/j.toxicon.2015.10.009 0041-0101/© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 142 S. Itoi et al. / Toxicon 108 (2015) 141e146 (Hanifin et al., 2002; Cardall et al., 2004). These findings suggest extraction and the remaining eggs were stored at À30 C until TTX that TTX accumulates in pufferfish by means other than through extraction. the classical food chain; since in vivo cultured TTX-producing bacteria are unable to produce enough quantities of TTX to ac- 2.2. DNA extraction and PCR amplification count for the amount of TTX in wild pufferfish (Miyazawa and Noguchi, 2001; Food Safety Commission of Japan, 2005; Wu et al., Total genomic DNA was extracted from each egg by the method 2005; Wang et al., 2008; Yang et al., 2010). This also indicates of Sezaki et al. (1999). The fragments of partial mitochondrial DNA that TTX-bearing pufferfish prey are abundant. In any case, the were amplified by PCR using two primer sets, 16S AR-L (forward: 50- origin of TTX and the acquisition process are both likely to vary CGCCT GTTTA TCAAA AACAT-30) and 16S BR-H (reverse: 50-CCGGT across different animal species, and it remains unclear exactly CTGAA CTCAG ATCAC GT-30) for the 16S rRNA gene (Palumbi et al., where pufferfish acquire the large quantities of TTX that they 1991), and TCytb-F1 (forward: 50-ACCTR TGGCG TGAAA AACCA possess. YCGTT GT-30) and TCytb-R1 (reverse: 50-CATYC GGTTT ACAAG Recently, our lab unexpectedly found numerous eggs among the ACCGR CGCTC TG-30) for the cytochrome b gene. Primers for cyto- intestinal contents of the pufferfish, Takifugu niphobles, and partial chrome b gene were designed based on the mitochondrial DNA mitochondrial DNA sequences from these eggs identified them to sequences from multiple species of the family Tetraodontidae. PCR be those of another pufferfish species, namely, Takifugu pardalis.We amplification was performed in a 20 ml reaction mixture containing have demonstrated the toxification process using cultured puffer- genomic DNA as a template, 1 unit ExTaq DNA polymerase (Takara fish Takifugu rubripes in this study by means of experimentally Bio, Shiga, Japan), 1.6 ml of 2.5 mM deoxynucleotide triphosphates reproducing the serendipitous finding of eggs in the pufferfish gut, (dNTP), 5 mlof5mM primers and 2 mlof10Â ExTaq DNA polymerase thus indicating that pufferfish toxification manifests from the buffer (Takara Bio). The thermal cycling program for the PCR con- accumulated TTX at relatively higher trophic levels in the food sisted of an initial denaturation at 95 C for 1 min followed by 35 chain. cycles of denaturation at 95 C for 10 s, annealing at 55 C for 30 s and extension at 72 C for 45 s. 2. Materials and methods 2.3. Direct sequencing and phylogenetic analyses 2.1. The intestinal contents of wild pufferfish Prior to the direct sequencing of the amplified product, the DNA Wild specimens of the pufferfish, T. niphobles (body weight: fragment was purified by chloroform extraction, followed by 60.3 ± 25.7 g and 48.9 ± 21.2 g for females and males, respectively; polyethylene glycol (PEG) 8000 precipitation and ethanol precipi- detailed in Table 1) were collected from coastal waters in Nagai, tation. Sequencing was performed for both strands using a 3130xl Kanagawa, Japan (35120N, 139360E), on 13 March 2012 (water genetic analyzer (Applied Biosystems, Foster, CA, USA) and a BigDye temperature: 13.9 C; salinity: 31.4 practical salinity unit, psu), 08 Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied March 2013 (16.0 C; 32.4 psu) and 14 March 2013 (14.2 C; 30.6 Biosystems). The concatenated nucleotide sequences of the 16S psu), 07 March 2014 (12.5 C; 32.3 psu) and 13 March 2014 (12.5 C; rRNA gene and cytochrome b gene of eggs were aligned using 31.8 psu), and 09 March 2015 (11.7 C; 34.3 psu), 16 March 2015 CLUSTAL W (Thompson et al., 1994) with those in the DDBJ/EMBL/ (12.3 C; 34.8 psu) and 19 March 2015 (14.6 C; 35.0 psu). The GenBank databases obtained using a BLAST search (Altschul et al., gonadosomatic index (GSI) was 2.2 ± 0.9 for females and 2.0 ± 1.4 1997). The alignment was then subjected to phylogenetic infer- for males (detailed in Table 1). The unexpected eggs that were ence by means of the maximum likelihood method using MEGA ver. found among the gut contents of the pufferfish were also collected. 6.0.6 (Tamura et al., 2013), with the corresponding concatenated Five eggs from each fish were immediately subjected to DNA sequences from the yellow-stripe toadfish, Torquigener brevipennis Table 1 Characteristics of the Takifugu niphobles specimens used in this studya. Sampling Sex No. of No. of egg-fed Intestinal egg content Toxicity of the intestinal eggs Standard length Body weight GSIb date specimen specimen (g)